Novel Nylanderia Pubens Virus

ABSTRACT

At least one novel virus capable of infecting crazy ants ( Nylanderia pubens ) is isolated, along with polynucleotide sequences and amino acid sequences of the virus(es). The virus(es) is capable of be used as a biopesticide to control populations of crazy ants.

CROSS REFERENCE TO RELATED APPLICATIONS:

This application claims priority to U.S. Patent Application 61/762,529filed Feb. 8, 2013.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to one or more novel viruses that infectNylanderia pubens. The invention also relates to polynucleotidessequences of these one or more novel viruses, to biopesticidescontaining these one or more novel viruses, and methods of using thebiopesticides.

2. Description of the Prior Art

Nylanderia pubens (Forel), previously Paratrechina pubens (LaPolla, etal., Syst. Entomol. 35:118-131 (2010)), is an invasive ant species thatin recent years has developed into a serious pest problem in theCaribbean and United States (see, e.g., Wetterer and Keularts, Entomol.91:423-427 (2008); and MacGown and Layton, Midsouth Entomol. 3:44-47(2010)). A rapidly expanding range, explosive localized populationgrowth, and control difficulties have elevated this ant to pest status.Professional entomologists and the pest control industry in the UnitedStates are urgently trying to understand its biology and developeffective control methods (see, e.g., Drees, et al., College Station:Texas A&M. 129-134 p. (2009)(insects.tamu.edu/fireant/research/projects/pdf/rasberrycrazyant.pdf);Warner and Scheffrahn, Gainesville: University of Florida, (2010)(edis.ifas.ufl.edu/pdffiles/IN/IN56000.pdf); Calibeo and Oi, ENY-2006(IN889) ed. Gainesville: University of Florida (2011)(edis.ifas.ufl.edu/pdffiles/IN/IN88900.pdf)). Efforts have primarilyfocused on pursuing development of insecticide-based control strategies(Meyers, College Station: Texas A&M. 163 p. (2008)(urbanentomology.tamu.edu/pdf/meyer_dissertation.pdf)), as well as theeffort to identify self-sustaining, biological control agents specificto N. pubens. While viruses can be important biological control agentsagainst pest insect populations (Lacey, et al., Biol. Cont. 21:230-248(2001)), none are known to infect N. pubens.

There remains a need for biocontrol agents and/or biopesticides thateliminate or at least reduce the spread of N. pubens and their colonies.The present invention is directed to one or more novel N. pubensviruses, polynucleotides of the one or more novel viruses, biopesticidescontaining the one or more novel N. pubens viruses, and methods of usingthe biopesticides to control the N. pubens population in an area.

BRIEF DESCRIPTION OF THE INVENTION

It is an object of this invention to have at least one novel virus thatinfects Nylanderia pubens and other types of crazy ants.

It is an object of this invention to have a biopesticide containing atleast one novel virus that infects Nylanderia pubens and other types ofcrazy ants.

It is another object of this invention to have a biopesticide containingat least one novel virus that infects Nylanderia pubens and other typesof crazy ants. It is a further object of this invention that thebiopesticide contains a carrier. The carrier can be a liquid or solid.It is another object of this invention that the carrier be a food sourcefor the crazy ants.

It is an object of this invention to have novel polynucleotide sequencesthat is SEQ ID NOs: 1, 2 and 3; has at least 95% identity to SEQ ID NO:1, SEQ ID NO: 2, or SEQ ID NO: 3; has at least 90% identity to SEQ IDNO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; or has at least 85% identity toSEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

It is an object of this invention to have novel polynucleotidesequences, SEQ ID NOs: 1, 2 and 3; has at least 95% identity to SEQ IDNO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; has at least 90% identity to SEQID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; or has at least 85% identity toSEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. It is a further object ofthis invention that these novel polynucleotide sequences, the RNAequivalent of these sequences, or the RNA equivalent of the complementof these sequences are viral genomic sequences. It is another object ofthis invention that the virus infects crazy ant.

It is another object of this invention that the genome of at least onenovel virus contains at least one of SEQ ID NOs: 1, 2 and 3; at leastone RNA equivalent of these sequences; at least one RNA equivalent ofthe complement of SEQ ID NOs: 1, 2 and 3; or a sequence that has atleast 95%, 90% or 85% identity to these sequences.

It is an object of this invention to have novel polypeptides that areencoded by the novel polynucleotide sequences, SEQ ID NOs: 1, 2 and 3;the complement of SEQ ID NOs: 1, 2, and 3; the reverse of SEQ ID NOs: 1,2 and 3; or the reverse complement of SEQ ID NOs: 1, 2 and 3; or asequence that has at least 95%, 90% or 85% identity to these sequences.

It is an object of this invention to have novel polypeptides that areencoded by the novel polynucleotide sequences, SEQ ID NOs: 1, 2 and 3;the complement of SEQ ID NOs: 1, 2, and 3; the reverse of SEQ ID NOs: 1,2 and 3; or the reverse complement of SEQ ID NOs: 1, 2 and 3; or asequence that has at least 95%, 90% or 85% identity to these sequences.It is another object of this invention that at least one virus containsat least one of these polypeptides. It is a further object of theinvention that the virus containing at least one of these polypeptidesinfects crazy ants.

It is an object of this invention to have a polypeptide encoded by thepolynucleotide sequences of SEQ ID NOs: 1, 2 or 3; the complement of SEQID NOs: 1, 2 or 3; the reverse of SEQ ID NOs: 1, 2, or 3; or thecomplement of the reverse of SEQ ID NOs: 1, 2 or 3; or a sequence thathas at least 95%, 90% or 85% identity to these sequences. It is anotherobject of this invention to have a virus containing a polypeptideencoded by the polynucleotide sequences of SEQ ID NOs. 1, 2 or 3; thecomplement of SEQ ID NOs: 1, 2 or 3; the reverse of SEQ ID NOs: 1, 2, or3; or the complement of the reverse of SEQ ID NOs: 1, 2 or 3; or asequence that has at least 95%, 90% or 85% identity to these sequences.It is a further object of this invention that the virus infects crazyants. It is another object of this invention to have a biopesticidecontaining this virus. The biopesticide can optionally contain acarrier. The carrier can be a solid or a liquid. Optionally the carrieris a food source for crazy ants.

It is another object of this invention to have a biopesticide containinga virus that infects crazy ants where the virus contains at least one ofthe polynucleotide sequences set forth in SEQ ID NOs: 1, 2 and 3, theRNA equivalent of SEQ ID NOs: 1, 2 and 3, or the RNA equivalent of thecomplement of SEQ ID NOs: 1, 2 and 3; or a sequence that has at least95%, 90% or 85% identity to these sequences. It is also an object ofthis invention that the biopesticide sickens and/or kills crazy ants. Itis a further object of this invention that the biopesticide contains acarrier where the carrier can be a food of crazy ants or a substancethat eases distribution or application of the biopesticide.

It is another object of this invention to have a biopesticide containinga virus that infects crazy ants where the virus contains at least one ofthe polynucleotide sequences set forth in SEQ ID NOs: 1, 2 and 3, theRNA equivalent of SEQ ID NOs: 1, 2 and 3, or the RNA equivalent of thecomplement of SEQ ID NOs: 1, 2 and 3; or a sequence that has at least95%, 90% or 85% identity to these sequences. It is also an object ofthis invention that the biopesticide sickens and/or kills crazy ants. Itis a further object of this invention that the biopesticide contains acarrier where the carrier can be a food of crazy ants or a substancethat eases distribution or application of the biopesticide. It isanother object of this invention that the carrier be either a solid or aliquid.

It is an object of this invention to have at least one virus that isidentifiable by at least one primer having a sequence of SEQ ID NOs: 4,5, 6, 7, 8, or 9 or mixtures thereof. It is a further object of thisinvention that the virus infects crazy ants.

It is another object of this invention to have a biopesticide containingat least one virus that is identifiable by at least one primer having asequence of SEQ ID NOs: 4, 5, 6, 7, 8, or 9 or mixtures thereof. It is afurther object of this invention that the virus infects crazy ants. Itis another object of this invention that the biopesticide optionallycontains a carrier. The carrier can optionally be a solid or a liquid.It is a further object of this invention that the carrier be a feedsource for the crazy ants.

It is another object of this invention to have a method for reducing thepopulation of crazy ants in a colony or eradicating a colony of crazyants by spreading a biopesticide in areas around a crazy ant colony orin areas where the crazy ants feed. It is a further object of thisinvention that the biopesticide contains at least one virus that infectscrazy ants. It is another object of this invention that the at least onevirus contains at least one of SEQ ID NOs: 1, 2 and 3, at least one RNAequivalent of these sequences, or at least one RNA equivalent of thecomplement of SEQ ID NOs: 1, 2 and 3; or a sequence that has at least95%, 90% or 85% identity to these sequences.

It is another object of this invention to have a method for reducing thepopulation of crazy ants in a colony or eradicating a colony of crazyants by spreading a biopesticide in areas around a crazy ant colony orin areas where the crazy ants feed. It is a further object of thisinvention that the biopesticide contains at least one virus that infectscrazy ants. It is another object of this invention that the at least onevirus have a polypeptide encoded by the polynucleotide sequences of SEQID NOs: 1, 2 or 3; the complement of SEQ ID NOs: 1, 2 or 3; the reverseof SEQ ID NOs: 1, 2, or 3; or the complement of the reverse of SEQ IDNOs: 1, 2 or 3; or a sequence that has at least 95%, 90% or 85% identityto these sequences.

It is another object of this invention to have a method for reducing thepopulation of crazy ants in a colony or eradicating a colony of crazyants by spreading a biopesticide in areas around a crazy ant colony orin areas where the crazy ants feed. It is a further object of thisinvention that the biopesticide contains at least one virus that infectscrazy ants. It is another object of this invention that the at least onevirus is identifiable by at least one primer having a sequence from thegroup of SEQ ID NOs: 4, 5, 6, 7, 8, or 9 or mixtures thereof.

DETAILED DESCRIPTION OF THE INVENTION

N. pubens is a species of ants formerly called Paratrechina pubens andis commonly called “crazy ants”. Crazy ants can include brown crazyants, tawny crazy ants, hairy crazy ants, Caribbean crazy ants, andRasberry crazy ants. Any hybrid ants that are infected by the virus ofthis invention are included in the term “crazy ants”. Crazy ants are aninvasive species that are a serious pest in the Caribbean and UnitedStates. Chemical pesticides may kill crazy ants but can also kill otherbeneficial or desired insects and animals. To date, no virus or bacteriahas been identified which infects crazy ants and which can be used as abiopesticide against crazy ants. This invention identifies at least onevirus that infects N. pubens, NpuV, and can be used as a biopesticideagainst crazy ants.

A “biocontrol agent” or “biopesticide” are interchangeable terms and arebroadly defined as a composition containing a protein, glycoprotein,polysaccharide, lipid, or other substance produced by animals, plants,bacteria, viruses, phages, fungi, protozoa, etc., that, when a pestingests, touches, or otherwise comes in contact with the composition,exerts a deleterious effect on the pest. Such deleterious effect caninclude, but is not limited to, inhibiting reproduction and/or killingthe pest. Viruses, bacteria, phages, protozoa, fungi, etc., can bebiocontrol agents or biopesticides in that these organisms can infectthe pest, injure, and/or kill the pest. Further, some animals arebiocontrol agents or biopesticides, such as endoparasitic wasps. In thisinvention, the at least one virus described herein, NpuV, can be abiocontrol agent or biopesticide for the crazy ants.

A biopesticide can optionally include a carrier component which can be aliquid, gel, or a solid material. The carrier usually is an inert agentthat does not repel the pest. The carrier may assist in the delivery ofthe biocontrol agent that targets the pest. A carrier can be a liquid,such as, but not limited to, water, sugar water, saline solution, oil,or any other liquid that does not adversely affect the viability and/oractivity of the biocontrol organism or compound. A solid carrier can be,for example, the pest's food or a substance that assists with theapplication or distribution of the biocontrol agent. For crazy ants,non-limiting examples of solid carriers include corn cob grits, extrudedcorn pellets, boiled egg yolks, and frozen insects such as crickets.

Optionally, a chemical pesticide, insecticide, or synergists can beincluded in the biopesticide. Non-limiting examples of pesticides,insecticides, or synergists for this invention include, abamectin,dinotefuran, avermectins, chlorfenapyr, indoxacarb, metaflumizone,imidacloprid, fipronil, hydramethylon, sulfluramid, hexaflumuron,pyriproxyfen, methoprene, lufenuron, dimilin, chlorpyrifos, neem,azadiractin, boric acid, their active derivatives, and the like. Thesepesticides/insecticides act as stressor which may be required toinitiate replication of the biocontrol organism which, in turn, resultsin death of the pests.

An “effective amount” or “amount effective for” is the minimum amount ofa biocontrol agent to affect the desired effect on the organism targetedby the biocontrol agent. For this invention, an “effective amount” or“amount effective for” is the minimum amount of the virus(es) orcomposition containing the virus(es) needed to cause the death of crazyants. An effective amount of the virus(es) of this invention will infectand kill a sufficient number of crazy ants such that the colony isreduced in size as compared to a similar colony that is not treated, orsuch that the colony collapses completely thereby eradicating the crazyants. The precise amount needed may vary in accordance with theparticular virus used, the other components of the biopesticide, thecolony being treated, the environment in which the colony is located,and the environment before, during, and after application of thebiocontrol agent. The exact amount of virus needed per dose ofbiopesticide and/or the amount of biopesticide needed can be easilydetermined by one of ordinary skill in the art using the teachingspresented herein.

The present invention includes the method of using the virus(es) of thepresent invention to reduce or eradicate a population of crazy ants.These methods involve spreading, distributing, or administrating thevirus(es) of the present invention or the biopesticide of the presentinvention to crazy ants, their colonies, areas around their colonies,and/or areas where the crazy ants forage and obtain food. The amount ofbiopesticide used is an effective amount for killing crazy ants,reducing the size of the colony compared to an untreated colony, oreradicating the crazy ants and their colony.

The terms “isolated”, “purified”, or “biologically pure” as used herein,refer to material that is substantially or essentially free fromcomponents that normally accompany the material in its native state. Inan exemplary embodiment, purity and homogeneity are determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A nucleicacid that is the predominant species present in a preparation issubstantially purified. In an exemplary embodiment, the term “purified”denotes that a nucleic acid or protein gives rise to essentially oneband in an electrophoretic gel. An isolated or purified virus is a virusthat is separated from other viruses with which it is found in nature orfrom the virus' host. Typically, isolated nucleic acids or proteins orviruses have a level of purity expressed as a range. The lower end ofthe range of purity for the component is about 60%, about 70% or about80% and the upper end of the range of purity is about 70%, about 80%,about 90% or more than about 90%.

The term “nucleic acid” as used herein, refers to a polymer ofribonucleotides or deoxyribonucleotides. Typically, “nucleic acid”polymers occur in either single- or double-stranded form, but are alsoknown to form structures comprising three or more strands. The term“nucleic acid” includes naturally occurring nucleic acid polymers aswell as nucleic acids comprising known nucleotide analogs or modifiedbackbone residues or linkages, which are synthetic, naturally occurring,and non-naturally occurring, which have similar binding properties asthe reference nucleic acid, and which are metabolized in a mannersimilar to the reference nucleotides. Exemplary analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleicacids (PNAs). “DNA”, “RNA”, “polynucleotides”, “polynucleotidesequence”, “oligonucleotide”, “nucleotide”, “nucleic acid”, “nucleicacid molecule”, “nucleic acid sequence”, “nucleic acid fragment”, and“isolated nucleic acid fragment” are used interchangeably herein.

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyino sine residues (see e.g., Batzer et al.,Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem.260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes8:91-98(1994)).

In addition to the degenerate nature of the nucleotide codons whichencode amino acids, alterations in a polynucleotide that result in theproduction of a chemically equivalent amino acid at a given site, but donot affect the functional properties of the encoded polypeptide, arewell known in the art. Thus, a codon for the amino acid alanine, ahydrophobic amino acid, may be substituted by a codon encoding anotherless hydrophobic residue, such as glycine, or a more hydrophobicresidue, such as valine, leucine, or isoleucine. Similarly, changeswhich result in substitution of one negatively charged residue foranother, such as aspartic acid for glutamic acid, or one positivelycharged residue for another, such as lysine for arginine or histidine,can also be expected to produce a functionally equivalent protein orpolypeptide.

As used herein a nucleic acid “probe”, oligonucleotide “probe”, orsimply a “probe” refers to a nucleic acid capable of binding to a targetnucleic acid of complementary sequence through one or more types ofchemical bonds, usually through complementary base pairing, usuallythrough hydrogen bond formation. As used herein, a probe may includenatural (i.e., A, G, C, or T) or modified bases (e.g., 7-deazaguanosine,inosine, etc.). In addition, the bases in a probe may be joined by alinkage other than a phosphodiester bond, so long as it does notinterfere with hybridization. Thus, for example, probes may be peptidenucleic acids in which the constituent bases are joined by peptide bondsrather than phosphodiester linkages. It will be understood by one ofskill in the art that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. Probes may contain a label so that one candetermine if the probe is bound to the target sequence. By assaying forthe presence or absence of the probe, one can detect the presence orabsence of the select sequence or subsequence. A probe can be bound,either covalently, through a linker or a chemical bond, ornoncovalently, through ionic, van der Waals, electrostatic, or hydrogenbonds, to a label such that the presence of the probe may be detected bydetecting the presence of the label bound to the probe. In otherexemplary embodiments, probes are indirectly labeled e.g., with biotinto which a streptavidin complex may later bind.

The term “label” as used herein, refers to a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. Exemplary labels include ³²P, fluorescent dyes, electron-densereagents, enzymes (e.g., as commonly used in an ELISA), biotin,digoxigenin, or haptens and proteins for which antisera or monoclonalantibodies are available. In one exemplary embodiment, labels can beisotopes, chromophores, lumiphores, chromogens, etc. Labels can alsoinvolve two or more compounds, only one of which need be attached to theprobe. An example of a pair of compounds that are labels is biotin andstreptavidin, where biotin is attached to the probe and later reactswith streptavidin which is added after the probe binds the targetsequence.

The term “primer” as used herein, refers to short nucleic acids,typically a DNA oligonucleotide of at least about 15 nucleotides inlength. In an exemplary embodiment, primers are annealed to acomplementary target DNA or RNA strand by nucleic acid hybridization toform a hybrid between the primer and the target DNA or RNA strand.Annealed primers are then extended along the target strand by a DNApolymerase enzyme or reverse transcriptase. Primer pairs can be used foramplification of a nucleic acid sequence, e.g., by the polymerase chainreaction (PCR) or other nucleic-acid amplification methods known in theart.

PCR primer pairs are typically derived from a known sequence, forexample, by using computer programs intended for that purpose such asPrimer (Version 0.5 ©1991, Whitehead Institute for Biomedical Research,Cambridge, Mass.). One of ordinary skill in the art will appreciate thatthe specificity of a particular probe or primer increases with itslength. Thus, for example, a primer comprising 20 consecutivenucleotides of a promoter complex sequence will anneal to a relatedtarget sequence with a higher specificity than a corresponding primer ofonly 15 nucleotides. Thus, in an exemplary embodiment, greaterspecificity of a nucleic acid primer or probe is attained with probesand primers selected to comprise 20, 25, 30, 35, 40, 50 or moreconsecutive nucleotides of a selected sequence.

Nucleic acid probes and primers are readily prepared based on thenucleic acid sequences disclosed herein. Methods for preparing and usingprobes and primers and for labeling and guidance in the choice of labelsappropriate for various purposes are discussed, e.g., in Green andSambrook, Molecular Cloning, A Laboratory Manual 4th ed. 2012, ColdSpring Harbor Laboratory; and Ausubel et al., eds., Current Protocols inMolecular Biology, 1994—current, John Wiley & Sons). The term“recombinant” when used with reference, e.g., to a cell, or nucleicacid, protein, or vector, indicates that the cell, organism, nucleicacid, protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant or wild-type) form of thecell or express native genes that are otherwise abnormally expressed,over expressed, under expressed or not expressed at all.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: “referencesequence”, “comparison window”, “sequence identity”, “percentage ofsequence identity”, and “substantial identity”. A reference sequence isa defined sequence used as a basis for a sequence comparison; areference sequence may be a subset of a larger sequence, or genesequence given in a sequence listing.

The terms “identical” or percent “identity”, in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(e.g., 85% identity, 90% identity, 99%, or 100% identity), when comparedand aligned for maximum correspondence over a comparison window, ordesignated region as measured using a sequence comparison algorithm orby manual alignment and visual inspection.

The phrase “substantially identical”, in the context of two nucleicacids or polypeptides, refers to two or more sequences or subsequencesthat have at least about 85%, identity, at least about 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%nucleotide or amino acid residue identity, when compared and aligned formaximum correspondence, as measured using a sequence comparisonalgorithm or by visual inspection. In an exemplary embodiment, thesubstantial identity exists over a region of the sequences that is atleast about 50 residues in length. In another exemplary embodiment, thesubstantial identity exists over a region of the sequences that is atleast about 100 residues in length. In still another exemplaryembodiment, the substantial identity exists over a region of thesequences that is at least about 150 residues or more, in length. In oneexemplary embodiment, the sequences are substantially identical over theentire length of nucleic acid or protein sequence.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from about 20 to about 600, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection (see, e.g.,Ausubel et al., eds., Current Protocols in Molecular Biology, 1995supplement).

An exemplary algorithm for sequence comparison is PILEUP. PILEUP createsa multiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity. It also plots a tree or dendogram showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360 (1987). The method used is similar to themethod described by Higgins & Sharp, CABIOS 5:151-153 (1989). Theprogram can align up to 300 sequences, each of a maximum length of 5,000nucleotides or amino acids. The multiple alignment procedure begins withthe pairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal., Nuc. Acids Res. 12:387-395 (1984)).

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., 1990). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The phrase “selectively hybridizes to” or “specifically hybridizes to”refers to the binding, duplexing, or hybridizing of a molecule only to aparticular nucleotide sequence under stringent hybridization conditionswhen that sequence is present in a complex mixture (e.g., total cellularor library DNA or RNA). In general, two nucleic acid sequences are saidto be “substantially identical” when the two molecules or theircomplements selectively or specifically hybridize to each other understringent conditions.

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Hybridization with Nucleic Probes Parts I andII, Elsevier (1993). Generally, stringent conditions are selected to beabout 5-10° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength pH. The T_(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g., 10 to50 nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For high stringencyhybridization, a positive signal is at least two times background,preferably 10 times background hybridization. Exemplary high stringencyor stringent hybridization conditions include: 50% formamide, 5×SSC and1% SDS incubated at 42° C. or 5×SSC and 1% SDS incubated at 65° C., witha wash in 0.2×SSC and 0.1% SDS at 65° C. However, other high stringencyhybridization conditions known in the art can be used.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides thatthey encode are substantially identical. This situation can occur, forexample, when a copy of a nucleic acid is created using the maximumcodon degeneracy permitted by the genetic code. In such cases, thenucleic acids typically hybridize under moderately stringenthybridization conditions. Exemplary “moderately stringent hybridizationconditions” include hybridization in a buffer of 40% formamide, 1 MNaCl, 1% SDS at 37° C., and a wash in 1×SSC at 45° C. A positivehybridization is at least twice background. Those of ordinary skill willreadily recognize that alternative hybridization and wash conditions canbe utilized to provide conditions of similar stringency.

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). Estimates are typically derived from agarose or acrylamidegel electrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides and polynucleotides that are not commercially availablecan be chemically synthesized e.g., according to the solid phasephosphoramidite triester method first described by Beaucage andCaruthers, Tetrahedron Letts. 22:1859-1862 (1981), or using an automatedsynthesizer, as described in Van Devanter et al., Nucleic Acids Res.12:6159-6168 (1984). Other methods for synthesizing oligonucleotides andpolynucleotides are known in the art. Purification of oligonucleotidesis by either native acrylamide gel electrophoresis or by anion-exchangeHPLC as described in Pearson & Reanier, J. Chrom. 255:137-149 (1983).

The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21-26(1981). Using of machines for sequencing DNA or RNA is known in the artfield.

This invention utilizes routine techniques in the field of molecularbiology. Basic texts disclosing the general methods of use in thisinvention include Green and Sambrook, 4th ed. 2012, Cold Spring HarborLaboratory; Kriegler, Gene Transfer and Expression: A Laboratory Manual(1993); and Ausubel et al., eds., Current Protocols in MolecularBiology, 1994—current, John Wiley & Sons. Unless otherwise noted,technical terms are used according to conventional usage. Definitions ofcommon terms in molecular biology maybe found in e.g., Benjamin Lewin,Genes IX, published by Oxford University Press, 2007 (ISBN 0763740632);Krebs, et al. (eds.), The Encyclopedia of Molecular Biology, publishedby Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

For all examples described herein, unless otherwise stated, the crazyants colonies are obtained from field sites located in Desoto (April,2011), Hillsborough (April, 2011), Alachua (March through May, 2011),and Duval (March, 2011) counties in Florida and subsequently aremaintained in the laboratory. Colonies are reared separately in nestingtubes as described by Oi and Williams, Environ. Entomol. 32:1171-1176(2003). The colonies are fed frozen crickets, 10% sucrose solution, andwater. Identifications of crazy ants are made based on characters listedin Trager (Sociobiol. 9:51-162 (1984)) and LaPolla, et al. (Syst Entomol35: 118-131 (2010)). While there is some uncertainty regarding speciesassignment of the crazy ants from Florida, Texas, Louisiana andMississippi based on morphometric and DNA sequence data, all of thesetypes of arts are considered crazy ants for the purpose of thisinvention.

Having now generally described this invention, the same will be betterunderstood by reference to certain specific examples and theaccompanying drawings, which are included herein only to furtherillustrate the invention and are not intended to limit the scope of theinvention as defined by the claims. The examples and drawings describeat least one, but not all embodiments, of the inventions claimed.Indeed, these inventions may be embodied in many different forms andshould not be construed as limited to the embodiments set forth herein;rather, these embodiments are provided so that this disclosure willsatisfy applicable legal requirements.

EXAMPLE 1 cDNA Library Generation and Analysis

Total RNA is extracted from samples of the colonies of N. pubens usingTRIzol® RNA isolation reagents (Life Technologies, Carlsbad, Calif.)according to the manufacturer's instructions. Samples are taken fromnine colonies. A total of 609 ants of different life stages (workers,alates, queens, larvae, pupae, and eggs) are used to prepare the totalRNA. RNA quality of each preparation is assessed by microfluidicanalysis on an Agilent 2100 Bioanalyzer (Agilent, Cary, N.C.) using theRNA 6000 Nano kit (Agilent, Cary, N.C.) according to the manufacturer'sdirections. Microfluidic assays are completed immediately after RNAextraction using a 1 μl volume of purified sample. RNA samples ofacceptable quality are pooled and are used as source material for mRNApurification. mRNA is isolated from the total RNA sample using theOligotex mRNA Mini Kit (Qiagen, Valencia, Calif.) following themanufacturer's instructions. The isolated mRNA is then utilized toprepare a non-normalized fragment library suitable for 454 platformsequencing using the NEBNext mRNA Sample Pre Reagent Set 2 (New EnglandBioLabs, Ipswich, Mass.) following the manufacturer's protocol. Thelibrary is used as a template for emulsion PCR using the GS Titanium LVemulsion PCR Kit (Lib-L; Roche, Basel, Switzerland) following themanufacturer's instructions. DNA beads generated from the emulsion PCRreactions are used for Titanium plate 454 sequencing, using the GSTitanium Sequencing Kit XLR70 (Roche, Basel, Switzerland). De novoassembly is performed for the generated sequencing data using theNewbler software (Roche).

The initial assembly of the sequences is performed with NewblerAssembler Version 2.3 (454 Life Science, Branford, Conn.), employingmasking and trimming sequencing repeats, primers and/or adaptors used incDNA library preparation. The hybridized sequences (contigs and leftoversingletons) are further assembled with Paracel Transcript Assemblerversion 3.0.0 (PTA; Paracel Inc., Pasadena, Calif.).

In PTA, all sequences are masked for universal and species-specificvector sequences, adaptors, and PCR primers used in cDNA librarycreation. Escherichia coli contamination and mitochondrial and ribosomalRNA genes are identified and are removed from input sequences usingdefault settings to ascertain the novelty of the sequences. The poly(A/T) tails and intrinsic repeats, such as simple sequence repeats andshort interspersed elements (SINE), are annotated prior to clusteringand assembly. Low base-call quality data are trimmed from the ends ofindividual sequences and sequences <75 by are excluded fromconsideration during initial pair-wise comparisons. After cleanup,sequences are passed to the PTA clustering module for pair-wisecomparison and then to the CAP3-based PTA assembly module for assembly.The PTA assembly is performed based on the sequences of the contigs andthe leftover singletons generated from the Newbler assembly.

The single production GS-FLX Titanium 454 platform sequencing run (twohalf plates) of the non-normalized N. pubens expression librarygenerates 1,306,177 raw sequence reads comprising 450 Mbp. De novoassembly of the raw data with Newbler yields 22,044 contigs and 232,338singletons. Subsequent assembly by PTA results in the generation of59,017 non-redundant sequences, including 27,348 contigs (average size794 bp) and 31,669 singlets (average size 295 bp). Among thesesequences, 27.9% (16,458) are greater than 500 by and 72.1% (42,533) aregreater than 300 bp. BLASTX analysis of these non-redundant nucleotidesequences identifies 25,898 (43.9%) nucleotide sequences withsignificant (e-value ≦1e⁻⁴) similarity, and 33,119 (56.1%) nucleotidesequences have no significant similarity. A significant percentage (47%)of the gene sequences (12,174) identified is found to be unique to N.pubens.

Large-scale homology database searches of the PTA sequence data set areconducted against the National Center for Biotechnology Information(NCBI) NR and NT databases using BLAST (blastx and blastn) with anin-house computational pipeline. To obtain a more accurate and completedescription of potential gene function for each queried sequence, thetop 100 BLAST hits are retrieved. Sequences with the best scoring BLASThit (≦1e⁻⁵) and the corresponding gene ontology (GO) classification areannotated to the queried sequence (Koski and Golding, J. Molec. Evol.52:540-542 (2001)). GO term assignments are binned according to thecategories, biological processes, cellular components, and molecularfunctions. BLAST results and GO term assignments are completed inBlastQuest, an SQL database developed by the Interdisciplinary Centerfor Biotechnology Research, University of Florida, that facilitatessimilarity-based sequence annotation with gene ontology information(Farmerie, et al., Data Know Eng. 53:75-97 (2005)). In addition, thesequences are characterized with respect to functionally annotated genesby BLAST searching against NCBI specific reference sequences (RefSeq)for Homo sapiens (38,556 sequences), Drosophila (21,099 sequences) andFormicidae (74,540 sequences). Queries are considered to have a clearhomolog of the searched organism when e-values are ≦1e⁻⁴, the length ofthe aligned segment is ≧50 bp, and identity >85%, which essentiallyeliminates spurious hits while preventing elimination of medium-sizedproteins.

Raw 454 reads and assembled contigs are deposited in the NCBI database.The N. pubens sequence data are publicly available and accessiblethrough the NCBI website accession numbers Ant_(—)454Assem_NCBI.sqn:JP773711-JP820231.

EXAMPLE 2 Identification of Viral Sequences

Sequences identified as exhibiting significant viral homology/identityare selected and are further evaluated in an attempt to establish theirorigin, viral, host, or otherwise. Evaluations are also conducted toascertain whether identified viral sequences are simply being ingestedby the ants or are replicating (i.e., N. pubens is serving as host).BLAST analysis of the 59,017 non-redundant sequences yielded from the N.pubens library results in the identification of 51 sequences of putativeviral origin. Among them, 31 sequences do not meet the threshold forsignificance (an expectation score >1e⁻⁴) and are not examined furtherto establish their source; viral, host, or otherwise. However, despiteexpectation scores greater than 1e⁻⁴, some of these sequences couldrepresent viruses that infect N. pubens.

Twenty sequences from the N. pubens expression library yield significantBLAST expectation scores of putative viral origin; nine sequences aresimilar to DNA virus sequences and eleven to RNA virus sequences. Of theeleven sequences related to genes of RNA viruses, six negative-sense andfive positive-sense, single-stranded RNA virus genes are identified.Three sequences of ostensibly positive-sense, single-stranded RNAvirus(es) are examined in more detail.

EXAMPLE 3 Confirmation of Viral Sequences

To confirm that the suspected RNA virus sequences are actually from RNAviruses, the forward and reverse oligonucleotide primers listed in Table1 are designed based on EST Assem.3776.C1 (GenBank Accession No.JP780688.1; SEQ ID NO: 1), EST Assem.13287.C1 (GenBank Accession No.JP790645.1; SEQ ID NO: 2), and EST Assem.8702.C1 (GenBank Accession No.JP786492.1; SEQ ID NO: 3).

TABLE 1 EST Forward oligonucleotide primer name, sequenceReverse oligonucleotide primer name, Designation (5′→3′), and SEQ ID NO.sequence (5′→3′), and SEQ ID NO. Assem.3776.C1 p1167 p1168 SEQ ID NO: 1CCCTACTGACTGACGAACAGATTGCTTC TGTTGTTGAGCGTAATGAGTCCGTCCT SEQ ID NO: 4SEQ ID NO: 5 Assem.13287.C1 p1169 p1170 SEQ ID NO: 2ACTTCACTTGTATATGGAGATCCCTCCAT TTGCTTCGTGATATGTCATTCCTGGATA ACAA CAATSEQ ID NO: 6 SEQ ID NO: 7 Assem.8702.C1 p1172 p1171 SEQ ID NO: 3TGGTACTGGTATGTCGGATGTGATGAGCT TGAGGTCTTGACACTGGTAGTGTTGAA SEQ ID NO: 8ATGA SEQ ID NO: 9

PCR is conducted with RNase-treated DNA extracted from the same N.pubens colonies used in expression library creation in Example 1. Noamplicon is generated indicating that the sequences are not N. pubensgenomic sequences. Next, mRNA is isolated from N. pubens colonies as perthe protocol in Example 1 and the primers in Table 1 are used togenerate amplicons using RT-PCR. Two-step RT-PCR is employed to amplifya portion of the genome strand. First, 1 μl (50 ng) of total RNA ismixed with 10 mM dNTPs and 1 μM of the appropriate taggedoligonucleotide primer, is heated to 65° C. for 5 minutes, and then isplaced on ice for at least 1 minute. First strand buffer and Superscriptreverse transcriptase (RT, Invitrogen, Carlsbad, Calif.) are then added,and the reaction mixture is incubated at 55° C. for 1 hour beforeinactivating the RT at 70° C. for 15 minutes. Unincorporatedoligonucleotides are digested with 10 units of Exonuclease I (NewEngland Biolabs, Ipswich, Mass.) at 37° C. for 1 hour. The reaction isterminated by heating to 80° C. for 20 minutes. Amplification isobserved in approximately 50% to approximately 66% of the samples, thusit is assumed that the sequences are not host (or other) origin, becauseviral infections rarely exhibit an incidence of 100% amongfield-collected arthropods (Fuxa and Tanada, Epizootiology of insectdiseases. New York: John Wiley and Sons. (1987)).

Next, tagged-RT-PCR is conducted with the above indicated primers todetect the replicating genomic strand (see Craggs, et al., J. Virol.Methods 94:111-120 (2001)). This method permits discrimination of eachgenome strand without carryover effects causing false positive detectionof either strand. Tagged-RT-PCR employs the use of the appropriateoligonucleotide primer in Table 1 appended at the 5′ end with a TAGsequence (5′-GGCCGTCATGGTGGCGAATAA-3′; SEQ ID NO: 10) that is used in acDNA-synthesis reaction (forward primer for positive-strand viruses andreverse primer for negative-strand viruses).

PCR is subsequently conducted in a 25 μl volume containing 2 mM MgCl₂,200 μM dNTP mix, 0.5 units of Platinum Taq DNA polymerase (Invitrogen,Carlsbad, Calif.), 0.2 μM of each oligonucleotide primer, and 5 μl ofthe cDNA preparation. PCR is conducted with the following temperatureregime, 94° C. for 2 minutes followed by 35 cycles of 94° C. for 15seconds, 56-60° C. for 15 seconds; 68° C. for 30 seconds and a final 68°C. step for 5 minutes in a thermal cycler. PCR products are separated ona 1% agarose gel and are visualized by SYBR-safe (Invitrogen, Carlsbad,Calif.) staining. The replicating strand for each of these ESTs aredetected by tagged-PCR which indicates that N. pubens is a host for thevirus. Next, the amplicon is cloned by ligating into the pCR4 expressionvector and transforming the pCR4 expression vector into TOP10 competentcells (Invitrogen, Carlsbad, Calif.). Its sequence is verified by Sangersequencing which is performed by the University of Florida,Interdisciplinary Center for Biotechnology Research (Gainesville, Fla.).Thus, EST Assem.3776.C1, EST Assem.13287.C1, and EST Assem.8702.C1 areconsidered to be from at least one virus infecting N. pubens.

The sequences of Assem.13287.C1, Assem.8702.C1, and Assem.3776.C1exhibit some homology to unclassified positive-strand RNA viruses.Specifically, Assem.13287.C1 exhibits some homology with the helicaseregion (ATPase chaperone functionality) of Kelp fly virus (Hartley, etal., J. Virol. 79:13385-13398 (2005)), and Assem.8702.C1 exhibits somehomology with the 3C-like protease region of polyprotein 1 (5′-proximalORF) of Solenopsis invicta virus 3 (SINV-3) (Valles and Hashimoto,Virology 388:354-361 (2009)). Assem3776.C1 also exhibits some homologywith SINV-3 at the interface of the 3C-protease and RNA-dependent RNApolymerase. The replicative form of the genome (minus or negativestrand) is detected in a small percentage of field-collected N. pubensproviding strong evidence that these sequences correspond topositive-strand RNA viruses that infect N. pubens. All of thesesequences exhibit identity to viruses that infect arthropods (Dipteraand Hymenoptera). Thus, detection of Assem.13287.C1, Assem.8702.C1, andAssem.3776.C1 by tagged-RT-PCR among a small percentage offield-collected N. pubens colonies suggests that these sequences are ofviral origin and appear to replicate in N. pubens. Sanger sequencing ofthese amplicons verifies their identity.

The primers listed in Table 1 (SEQ ID NOs: 4, 5, 6, 7, 8, and 9) can beused to identify NpuV. The reverse primers (SEQ ID NOs: 5, 7, and 9) canhybridize to the RNA genome of NpuV and to a cDNA of the viral genome.The forward primers (SEQ ID NOs: 4, 6, and 8) hybridize to a negativeRNA strand of the viral genome and to a cDNA of the viral genome. NpuV,as a positive-strand RNA virus, typically contains only the positivestrand RNA in its capsid. But during active replication in infectedcells, the infected cells contain copies of both the positive-strand RNAand the negative-strand RNA which serves as template to make additionalcopies of the positive-strand RNA.

EXAMPLE 4 Characteristics of Viruses

The virus(es) of this invention, referred to as Nylanderia pubens virusor NpuV, or as NpuV-1, NpuV-2, and NpuV-3, represents the firstvirus(es) to be discovered from an ant in the Formicine. The virus(es)of this invention possesses features consistent with placement withinthe order Picornavirales: 1. non-enveloped particles with a diameteraround 30 nm, 2. a positive-sense, single-stranded RNA genome, 3. noproduction of subgenomic RNA, and 4. a polyprotein containing helicase,protease, and RdRp domains (Le Gall, et al., Archives of Virology 153,715-27 (2008)).

A series of 5′ RACE reactions are conducted to obtain the upstreamsequences of the genome of the virus(es) of this invention using the 5′RACE system (Invitrogen, Carlsbad, Calif.) and primer walking. For eachreaction, cDNA is synthesized for 50 minutes at 48° C. with 2.5 μg oftotal RNA extracted with TRIzol® RNA isolation reagents (LifeTechnologies, Carlsbad, Calif.) according to manufacturer's instructionsfrom Nylanderia pubens virus or Nylanderia pubens virus-infected antswith gene-specific oligonucleotide primer, the RNA template is degradedwith RNase H, and the cDNA purified. The 3′ end of the cDNA ispolycytidylated with terminal deoxynucleotidyl transferase and dCTP. Thetailed cDNA is then amplified with a nested GSP (3′ end) and an abridgedanchor primer (AAP). Gel-purified amplicons are ligated into thepCR4-TOPO vector, transformed into TOP10 competent cells (Invitrogen,Carlsbad, Calif.) and are sequenced by the Interdisciplinary Center forBiotechnology Research (University of Florida).

To obtain sequences downstream of the amplicon, 3′ RACE reactions areconducted with the GeneRacer kit (Invitrogen, Carlsbad, Calif.)according to manufacturer's instructions. cDNA is synthesized from totalRNA (1 μg) using the GeneRacer appended Oligo dT primer. The cDNA isamplified by PCR with a GSP and GeneRacer 3′ primer. Amplicons arecloned and sequenced as described for 5′ RACE.

EXAMPLE 5 Viral Transmission

To evaluate the transmission of the at least one of the viruses of thisinvention, uninfected crazy ant nests are identified by RT-PCR as perExample 1, excavated from the field, and parsed into equivalentcolonies. Colonies are infected by a modification of the methoddescribed by Ackey and Beck (J. Insect Physiol. 18:1901-1914 (1972)).Briefly, approximately 1 to approximately 5 grams of crazy ants from acolony infected with at least one of the viruses of this invention arehomogenized in a Waring blender with either 30 ml of 10% sucroseprepared with deionized water or 30 ml of refined soybean oil for 1minute at high speed. The homogenates are filtered through three layersof cheesecloth and then filtered by vacuum in a Buchner funnel through anumber 1 Whatman paper. The 10% sucrose-virus bait is used in this form.The oil-virus bait preparation is mixed with pregel defatted corn grit(Illinois Cereal Mills, Paris, Ill.) at 30% oil-virus by weight toadsorb the oil onto the corn grit. In addition, approximately 1 toapproximately 5 grams of infected crazy ants are mixed with 21 g offreeze-killed adult house crickets. The mixture is pulverized with amortar and pestle to create a crude paste. These three types of infectedfood sources are placed in a position that the uninfected crazy antcolony will find it and partake thereof. The control colony is providedthe same three food sources, but without the infected crazy ants addedto it. Crazy ants from the treated and untreated colonies are sampled atabout 3, 11 and 18 days after introduction of the food source (infectedor non-infected) and analyzed for the presence of at least one of theviruses of this invention by RT-PCR using the primers in Table 1 and theprotocol described above.

EXAMPLE 6 Biopesticide Efficacy

To evaluate the efficacy of using at least one of the viruses of thisinvention as a biopesticide, non-infected colonies are obtained andplaced in the laboratory. Homogenates of crazy ant colonies infectedwith at least one of the viruses of this invention are used as sourcesof virus for preparation of the bait formulations. Aliquots are blendedin a Waring blender with either 30 ml of 10% sucrose prepared withdeionized water or 30 ml of refined soybean oil for 1 minute at highspeed. The homogenates are filtered through three layers of cheeseclothand then filtered by vacuum in a Buchner funnel through a number 1Whatman paper. The 10% sucrose-virus bait is used in this form. Theoil-virus bait preparation is mixed with pregel defatted corn grit(Illinois Cereal Mills, Paris, Ill.) at 30% oil-virus by weight toadsorb the oil onto the corn grit. The third aliquot of infected crazyants is mixed with 21 g of freeze-killed adult house crickets. Themixture is pulverized with a mortar and pestle to create a crude paste.Virus content of each bait formulation is determined by quantitative PCRusing the primers in Table 1.

Test colonies (n=10/treatment) are transferred into clean trays withoutfood two days before test treatments and then pulse exposed to virusbait formulations for 24 hours. Control colonies receive access tocrickets, sugar water, and oil on corn grits, all without the virushomogenate. The baits are subsequently placed on the floor of the antrearing tray. Treatment and control colonies are randomly distributed inthe holding trays until virus replication was confirmed in thetreatments, after which, colonies positive for virus replication areremoved into separate holding trays in an adjacent rack to limit chancesfor contamination of uninfected colonies.

Bait-treated and control colonies are periodically evaluated toascertain their relative health by monitoring the ratio of brood toworkers. Colonies are assigned a score of 4 if they are rapidly growingwith brood mass greater than worker mass. A score of 3 indicates ahealthy growing colony with brood mass approximately 70% of worker mass.A score of 2 indicates a colony in poor health with brood mass ofapproximately 50% of worker mass. A score of 1 indicates a sick colonywith brood mass approximately 25% of worker mass. A score of 0 isreserved for colonies without any brood. During evaluations each nesttube is manually checked and intermediate scores are assigned asappropriate. Control colonies are always checked first and gloves werechanged between each colony to avoid mechanically transmitting thevirus. Each colony is evaluated to determine the quantity of workers (g)and brood (g), worker and larval mortality (number of dead workerscounted in the housing tray), the queen weight, queen ovary rating, thenumber of eggs laid by the queen in a 24 hour period.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation. Alldocuments cited herein are incorporated by reference.

1-2. (canceled)
 3. A biopesticide comprising an isolated virus thatinfects and kills crazy ants and a carrier; wherein said carriercomprises a food for crazy ants; and wherein said virus comprises a RNApolynucleotide having a sequence equivalent to the sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, asequence that has at least 95% identity to SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 3, a sequence that has at least 90% identity to SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, and a sequence that has at least 85%identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:
 3. 4. Thebiopesticide of claim 3 wherein said food is selected from the groupconsisting of corn grits, extruded corn pellets, boiled egg yolks,frozen insects, and a combination thereof.
 5. A method of reducing thepopulation of crazy ants in a colony or eradicating a colony of crazyants comprising administering, in an amount effective to kill crazyants, the biopesticide of claim 3 to areas around a crazy ant colony orto areas where the crazy ants feed. 6-7. (canceled)
 8. A biopesticidecomprising an isolated virus that infects and kills crazy ants and acarrier; wherein said carrier comprises a food for crazy ants; andwherein said virus comprises a polypeptide encoded by a polynucleotidehaving a DNA sequence of SEQ ID NO:
 1. 9. The biopesticide of claim 8wherein said food is selected from the group consisting of corn grits,extruded corn pellets, boiled egg yolks, frozen insects, and acombination thereof.
 10. A method of reducing the population of crazyants in a colony or eradicating a colony of crazy ants comprisingadministering, in an amount effective to kill crazy ants, thebiopesticide of claim 8 to areas around a crazy ant colony or to areaswhere the crazy ants feed.
 11. (canceled)
 12. A biopesticide comprisingan isolated virus that infects and kills crazy ants and a carrier,wherein said virus is identifiable by at least one primer selected fromthe group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and mixtures thereof; and whereinsaid carrier comprises a food for crazy ants.
 13. The biopesticide ofclaim 12 wherein said food is selected from the group consisting of corngrits, extruded corn pellets, boiled egg yolks, frozen insects, and acombination thereof.
 14. A method of reducing the population of crazyants in a colony or eradicating a colony of crazy ants comprisingadministering, in an amount effective to kill crazy ants, thebiopesticide of claim 12 to areas around a crazy ant colony or to areaswhere the crazy ants feed.
 15. The method of claim 5 wherein said foodis selected from the group consisting of corn grits, extruded cornpellets, boiled egg yolks, frozen insects, and a combination thereof.16. The method of claim 5 wherein said carrier further comprises aliquid selected from the group consisting of water, sugar water, salinesolution, and oil.
 17. The method of claim 10 wherein said food isselected from the group consisting of corn grits, extruded corn pellets,boiled egg yolks, frozen insects, and a combination thereof.
 18. Themethod of claim 10 wherein said carrier further comprises a liquidselected from the group consisting of water, sugar water, salinesolution, and oil.
 19. The method of claim 14 wherein said food isselected from the group consisting of corn grits, extruded corn pellets,boiled egg yolks, frozen insects, and a combination thereof.
 20. Themethod of claim 14 wherein said carrier further comprises a liquidselected from the group consisting of water, sugar water, salinesolution, and oil.